The Reprolysin Jararhagin, a Snake Venom Metalloproteinase, Functions as a Fibrillar Collagen Agonist Involved in Fibroblast Cell Adhesion and Signaling
2002; Elsevier BV; Volume: 277; Issue: 43 Linguagem: Inglês
10.1074/jbc.m202049200
ISSN1083-351X
AutoresPaola Zigrino, Aura S. Kamiguti, Johannes A. Eble, Claudia Drescher, Roswitha Nischt, Jay W. Fox, Cornelia Mauch,
Tópico(s)Protease and Inhibitor Mechanisms
ResumoThe integrins α2β1 and α1β1 have been shown to modulate cellular activities of fibroblasts on contact with fibrillar collagen. Previously it has been shown that collagen binding to α2β1 regulates matrix metalloproteinase MMP-1 and membrane-type MT1-MMP expression. Jararhagin is a snake venom metalloproteinase of the Reprolysin family of zinc metalloproteinases, containing a metalloproteinase domain followed by disintegrin-like and cysteine-rich domains. Jararhagin blocks type I collagen-induced platelet aggregation by binding to the α2β1integrin and inhibiting collagen-mediated intracellular signaling events. Here we present evidence that, in contrast to the observations in platelets, jararhagin binding to the integrin receptor α2β1 in fibroblasts produces collagen-like cell signaling events such as up-regulation of MMP-1 and MT1-MMP. Inactivation of the metalloproteinase domain had no effect on these properties of jararhagin. Thus, in fibroblasts the snake venom metalloproteinase jararhagin functions as a collagen-mimetic substrate that binds to and activates integrins. Given the homology between the metalloproteinase, disintegrin-like and cysteine-rich domains of jararhagin and those of the members of the ADAMs (adisintegrin-like andmetalloproteinase) family of proteins, this work demonstrates the potential of the disintegrin-like/cysteine-rich domains in the ADAMs as cellular signaling agents to elicit responses relevant to the biological function of these proteins. The integrins α2β1 and α1β1 have been shown to modulate cellular activities of fibroblasts on contact with fibrillar collagen. Previously it has been shown that collagen binding to α2β1 regulates matrix metalloproteinase MMP-1 and membrane-type MT1-MMP expression. Jararhagin is a snake venom metalloproteinase of the Reprolysin family of zinc metalloproteinases, containing a metalloproteinase domain followed by disintegrin-like and cysteine-rich domains. Jararhagin blocks type I collagen-induced platelet aggregation by binding to the α2β1integrin and inhibiting collagen-mediated intracellular signaling events. Here we present evidence that, in contrast to the observations in platelets, jararhagin binding to the integrin receptor α2β1 in fibroblasts produces collagen-like cell signaling events such as up-regulation of MMP-1 and MT1-MMP. Inactivation of the metalloproteinase domain had no effect on these properties of jararhagin. Thus, in fibroblasts the snake venom metalloproteinase jararhagin functions as a collagen-mimetic substrate that binds to and activates integrins. Given the homology between the metalloproteinase, disintegrin-like and cysteine-rich domains of jararhagin and those of the members of the ADAMs (adisintegrin-like andmetalloproteinase) family of proteins, this work demonstrates the potential of the disintegrin-like/cysteine-rich domains in the ADAMs as cellular signaling agents to elicit responses relevant to the biological function of these proteins. Adhesion of fibroblasts to native type I collagen is mediated by α1β1 and α2β1integrin receptors (1Hemler M.E. Ann. Rev. Immunol. 1990; 8: 365-400Google Scholar, 2Hynes R.O. Cell. 1992; 9: 11-25Google Scholar). Recently, Knight et al. (3Knight C.G. Morton L.F. Peachey A.R. Tuckwell D.S. Farndale R.W. Barnes M.J. J. Biol. Chem. 2000; 275: 35-40Google Scholar) have shown that the sequence GFOGER (O, hydroxyproline) in triple-helical collagen type I and IV is recognized by both α1β1 and α2β1integrins. Several studies have localized the binding site for collagen within the I-domain of the α-chain integrin subunit. The I-domain is composed of about 200 amino acids and shares homology with the von Willebrand factor A domain (4Hughes A.L. Mol. Biol. Evol. 1992; 9: 216-234Google Scholar, 5Eble J.A. Golbik R. Mann K. Kühn K. EMBO J. 1993; 12: 4795-4802Google Scholar, 6Tuckwell D.S. Calderwood D.A. Green L.J. Humphries M.J. J. Cell Sci. 1995; 108: 1629-1637Google Scholar). When human dermal fibroblasts are grown in contact with fibrillar collagen type I, a series of events are triggered. Fibroblasts acquire phenotypic tissue-like characteristics that are not observed in fibroblasts grown as monolayer cultures on plastic or on monomeric collagen type I (7Grinnel F. J. Cell Biol. 1994; 124: 401-404Google Scholar, 8Grinnel F., Ho, C.-H. Lin Y.-C. Skuta G. J. Biol. Chem. 1999; 274: 918-923Google Scholar). When seeded into these loose networks of collagen fibrils, fibroblasts down-regulate type I collagen expression (9Mauch C. Hatamochi A. Scharffetter K. Krieg T. Exp. Cell Res. 1988; 178: 493-503Google Scholar), induce MMP-1 1The abbreviations used are: MMP-1, matrix metalloproteinase-1; MMP-2, matrix metalloproteinase-2; MT1-MMP, membrane-type matrix metalloproteinase-1; PBS, phosphate-buffered saline; SVMP, snake venom metalloproteinases; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; BSA, bovine serum albumin. synthesis (10Mauch C. Adelmann-Grill C.B. Hatamochi A. Krieg T. FEBS Lett. 1989; 250: 301-305Google Scholar), and activate pro-MMP-2 (11Seltzer J.L. Lee A.-Y. Akers K.T. Sudbeck B. Southon E.A. Wayner E.A. Eisen A.Z. Exp. Cell Res. 1994; 213: 365-374Google Scholar). Furthermore, it has been shown in fibroblasts that collagen binding to the α2β1 integrin contributes to the reorganization and contraction of the collagenous matrix (12Chan B.M. Matsuura N. Takada Y. Zetter B.R. Hemler M.E. Science. 1991; 251: 1600-1602Google Scholar, 13Klein C.E. Dressel D. Steinmayer T. Mauch C. Eckes B. Krieg T. Bankert R.B. Weber L. J. Cell Biol. 1991; 115: 1427-1436Google Scholar) and is responsible for the induction of MMP-1 synthesis (14Langholz O. Roeckel D. Mauch C. Kozlowska E. Bank I. Krieg T. Eckes B. J. Cell Biol. 1995; 131: 1903-1915Google Scholar, 15Riikonen T. Westermarck J. Koivisto L. Broberg A. Kähäri V.-M. Heino J. J. Biol. Chem. 1995; 270: 13548-13552Google Scholar). The down-regulation of type I collagen synthesis in this system was due to collagen binding to the α1β1 integrin (14Langholz O. Roeckel D. Mauch C. Kozlowska E. Bank I. Krieg T. Eckes B. J. Cell Biol. 1995; 131: 1903-1915Google Scholar). Recently, we observed that, in addition to MMP-1, MT1-MMP is also induced on both the mRNA and protein levels by the ligation of the α2β1 integrin receptor with fibrillar collagen (16Zigrino P. Drescher C. Mauch C. Eur. J. Cell Biol. 2001; 80: 68-77Google Scholar). The synthesis of the α2β1integrin was found to be up-regulated in collagen lattices, whereas the expression of other collagen integrin receptors, such as α1β1 and α3β1, was not affected (13Klein C.E. Dressel D. Steinmayer T. Mauch C. Eckes B. Krieg T. Bankert R.B. Weber L. J. Cell Biol. 1991; 115: 1427-1436Google Scholar). The snake venom metalloproteinases (SVMPs) are members of the Reprolysin family (M13) of metalloproteinases. The ADAMs (adisintegrin-like andmetalloproteinase)/MDC (metalloproteinase,disintegrin, cysteine-rich) group of proteins are also members of the Reprolysin family (17Fox J.W. Long C. Bailey G. Snake Venom Enzymes. Alaken Press, Ft. Collins, CO1998: 151-178Google Scholar). The PIII class of SVMPs and the ADAMs share homologous metalloproteinase, disintegrin, and cysteine-rich domains (18Jia L.-G. Shimokawa K.-I. Bjarnason J.B. Fox J. Toxicon. 1996; 34: 1269-1276Google Scholar). Based on these similarities, the SVMPs have served as early models for ADAM function. The PIII SVMPs have been demonstrated to be capable of proteolytically degrading extracellular matrix and inhibiting platelet aggregation by blocking collagen binding to the α2β1 integrin on platelets (17Fox J.W. Long C. Bailey G. Snake Venom Enzymes. Alaken Press, Ft. Collins, CO1998: 151-178Google Scholar). Jararhagin, a hemorrhagic metalloproteinase from Bothrops jararaca, is one of the main venom components responsible for the local and systemic hemorrhage observed in envenomed humans (19Paine M.J.I. Desmond H.P. Theakston R.D.G. Crampton J.M. J. Biol. Chem. 1992; 267: 22869-22876Google Scholar). Jararhagin has been shown to degrade components of the basement membrane of the microvasculature and some plasma proteins important for hemostasis (20Kamiguti A.S. Hay C.R.M. Theakston R.D.G. Zuzel M. Toxicon. 1996; 34: 627-642Google Scholar, 21Hati R. Mitra P. Sarker S. Bhattacharyya K.K. Crit. Rev. Toxicol. 1999; 29: 1-19Google Scholar). Furthermore, it synergizes hemorrhage by inhibiting collagen-stimulated platelet aggregation (22Kamiguti A.S. Markland F.S. Zhou Q. Laing G.D. Theakston R.D.G. Zuzel M. J. Biol. Chem. 1997; 272: 32599-32605Google Scholar). In the venom, the metalloproteinase domain of jararhagin is often proteolytically processed generating jararhagin-C, a fragment representing the disintegrin and cysteine-rich domains of jararhagin. The α2β1 integrin can also interact with jararhagin-C, but the interaction seems to be weaker than with jararhagin (23De Luca M. Ward C.M. Ohmori K. Andrews R.K. Berndt M.C. Biochem. Biophys. Res. Commun. 1995; 206: 570-576Google Scholar), suggesting that additional N-terminal structures might be involved in jararhagin-α2β1 binding. Interestingly, synthetic peptides based on a sequence in the metalloproteinase domain of jararhagin have been shown to bind to the I-domain of the recombinant α2 integrin chain thereby preventing the binding of the α2-I domain to collagens I and IV, and to laminin-1 (24Ivaska J. Käpylä J. Pentikäinen O. Hoffren A.-M. Hermonen J. Huttunen P. Johnson M.S. Heino J. J. Biol. Chem. 1999; 274: 3513-3521Google Scholar). In this study, we have investigated the ability of jararhagin to mimic fibrillar collagen interaction with fibroblasts to modulate the expression of the integrin α2β1 and the matrix metalloproteinases MMP-1 and MT1-MMP. In contrast to previous studies performed in platelets, jararhagin binding to fibroblasts led to cellular activities similar to those induced by fibrillar type I collagen binding via the α2β1 integrin. These results suggest that other disintegrin-like/cysteine-rich domain-containing proteins, such as the ADAMs, may be capable of not only binding to integrins, as has been shown, but also signaling via integrins to alter cellular events such as gene and protein expression. The following antibodies were used: function blocking mouse monoclonal antibodies directed against the β1 (4B4; Coulter Corporation), α2 (P1E6; BIOMOL), and α3 (BIOMOL) integrin chains; monospecific mouse antibodies to MT1-MMP were raised against a peptide corresponding to the residues 160–173 of human MT1-MMP (114–1F2; Fuji Chemicals). Rabbit polyclonal antibodies to human MMP-1 were kindly provided by Dr. P. Angel (Deutsches Krebsforschungszentrum, Heidelberg, Germany). Function-blocking mouse antibodies raised against the human α1 integrin chain, mouse antibodies against human HLA-ABC, and antibodies directed against the β1 integrin subunit antibodies used for immunoblotting were from Chemicon. The rabbit polyclonal antibodies directed against jararhagin were a kind gift from Dr. R. D. G. Theakston (Liverpool School of Tropical Medicine, Liverpool, UK). Jararhagin was purified from the venom of B. jararaca as previously described (19Paine M.J.I. Desmond H.P. Theakston R.D.G. Crampton J.M. J. Biol. Chem. 1992; 267: 22869-22876Google Scholar). Inactivation of proteolytic activity was performed by a 5-min treatment with 5 mm 1,10-phenanthroline at 37 °C (25Kamiguti A.S. Hay C.R.M. Zuzel M. Biochem. J. 1996; 320: 635-641Google Scholar). Human dermal fibroblasts obtained by outgrowth from explants were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS), 2 mm glutamine, and 100 units/ml each of penicillin and streptomycin. Fibroblasts were used at passages 1–9. Three-dimensional collagen gels were prepared as described previously (9Mauch C. Hatamochi A. Scharffetter K. Krieg T. Exp. Cell Res. 1988; 178: 493-503Google Scholar). Briefly, type I collagen (3 mg/ml, Vitrogen) and 10× DMEM were combined in a 10:1 ratio and neutralized by the addition of 0.1 m NaOH. Fibroblasts were seeded into collagen gels at a density of 1 × 105 cells/ml and incubated at 37 °C. Alternatively, suspensions of fibroblasts were preincubated with jararhagin, 100 nm, for 20 min at 37 °C before seeding into collagen gels. Rates of gel contraction were monitored by determining the remaining surface area. For integrin binding experiments, cells were detached from confluent monolayer cultures by trypsinization, collected by centrifugation, and resuspended in growth medium. Before seeding onto tissue culture plastic plates, the cells were preincubated in the presence of different concentrations of antibodies for 20 min at 37 °C. Cell viability was determined by trypan blue exclusion. To detect α2 integrin, fibroblasts were cultured on chamber slides. After 48 h, cells were washed twice with phosphate-buffered saline (PBS) and then fixed with cold acetone. Staining was performed with rabbit anti-α2 antibodies against the cytoplasmic domain of the protein (Chemicon) overnight at 4 °C in PBS containing 4% BSA, followed by incubation with goat anti-rabbit-Cy3 antibodies (1:400 in PBS/2% BSA, Dianova) for 1 h at room temperature in a humidified chamber. Omission of the first antibody was used as a negative control. Semi-confluent fibroblast monolayer cultures were trypsinized, and the cells were washed with PBS and resuspended in DMEM containing 0.5% BSA and insulin, transferrin, and sodium selenite at concentrations as recommended by the manufacturer (Sigma). For competition assays, antibodies were added to the cell suspension before plating. Adhesion assays were performed as previously described (26Zigrino P. Gaietta G. Zambonin Zallone A. Pelletier A.J. Quaranta V. Biochim. Biophys. Res. Commun. 1996; 221: 51-58Google Scholar). Briefly, 96-well microtiter plates were coated with 100 μl of active or 1,10-phenanthroline-inactivated jararhagin (4 μg/ml), monomeric collagen type I (40 μg/ml), or 50% FCS at 4 °C overnight. BSA coating and blockage of nonspecific binding sites were performed by 1-h incubation with heat-denatured BSA (1% BSA in Ca2+/Mg2+-free PBS) at room temperature. After washing the wells twice, cells (2 × 104 cells/well) were seeded and incubated for 2 h at 37 °C. Non-adherent cells were removed by washing twice with PBS, and adherent cells were fixed with 3% formaldehyde in PBS, pH 7.6, and stained with 0.5% crystal violet in 20% (v/v) methanol. The dye was released from the cells by addition of 0.1 m sodium citrate in 50% (v/v) ethanol. The optical density of the released dye solution was determined at 595 nm. Values were calculated relative to the values obtained for the control assays (FCS or jararhagin pre-coated plates), which were arbitrarily set as 100%. Statistical analysis was performed with the ANOVA Dunnett multiple comparison test. Recombinant soluble human integrin α2β1 ectodomain heterodimers were prepared in insect cells using an expression plasmid in which the cytoplasmic and transmembrane domains were replaced by Fos and Jun dimerization motifs as described previously (27Eble J.A. Wucherpfennig K.W. Gauthier L. Dersch P. Krukonis E. Isberg R.R. Hemler M.E. Biochemistry. 1998; 37: 10945-10955Google Scholar). Microtiter plates were coated with jararhagin and bovine type I collagen at concentrations of 4 and 40 μg/ml in Tris-buffered saline (TBS, 50 mm Tris-HCl, pH 7.4, 150 mm NaCl) containing 3 mm MgCl2 and 0.1 macetic acid. After overnight incubation, the wells were blocked with heat-denatured BSA and incubated with 6 μg/ml soluble α2β1 integrin in the absence or presence of 10 mm EDTA for 2 h at room temperature. Then the wells were washed twice, and substrate-bound integrin was detected by enzyme-linked immunosorbent assay using a rabbit anti-human β1 integrin antiserum and alkaline phosphatase-coupled anti-rabbit IgG antibodies as primary and secondary antibodies, respectively. para-Nitrophenylphosphate was used as the enzyme-linked immunosorbent assay substrate with the product measured at 405 nm. Each value was measured in duplicate, and standard deviations were calculated. For preparation of crude plasma membranes, cells were washed twice with PBS and scraped off the plates with PBS containing the protease inhibitors aprotinin (10 μg/ml), Pefabloc (0.25 mg/ml), and leupeptin (1 μg/ml). Cell suspensions were subjected to three cycles of freeze-thaw in a dry ice-ethanol/37 °C bath, and cell lysis was confirmed microscopically. Lysates were separated from cell nuclei by centrifugation at 500 × g. Then the supernatant was centrifuged at 7,000 × g for 15 min at 4 °C. The crude plasma membranes were washed once with PBS/inhibitors and, after centrifugation, resuspended in PBS. For preparation of total lysates, cells were washed twice with PBS and lysed in PBS containing 0.5% Nonidet P-40. Lysates were centrifuged at 15,000 × gfor 20 min at 4 °C. Protein concentration was determined using a commercial assay (Bio-Rad). For Western blotting, equal amounts of protein from the membrane preparations, lysates, or conditioned media were separated on 10% SDS-polyacrylamide gels under reducing conditions and transferred onto Hybond-C Super™ (Amersham Biosciences). After blockage of nonspecific binding sites with 5% skimmed milk in PBS containing 0.5% Tween (v/v), the blots were incubated with the primary antibodies overnight at 4 °C. Bound primary antibodies were detected using a horseradish peroxidase-conjugated secondary antibody (1:2000, Dako) and visualized with the ECL system (ECL™, Amersham Biosciences). Purified jararhagin was biotin-conjugated with biotin-XX sulfosuccinimidyl ester. Labeling and purification of biotin-labeled jararhagin were performed using the FluoReporter® Mini-Biotin-XX protein labeling kit (Molecular Probes, Leiden, The Netherlands). The ability of jararhagin to displace bound, biotinylated jararhagin from fibroblasts was assayed by incubating fibroblasts in the presence of biotinylated jararhagin for 24 h followed by incubation with varying concentrations of unlabeled jararhagin for an additional 24 h. Cell surface binding of total jararhagin (jararhagin and biotinylated jararhagin) and biotinylated jararhagin after the 48 h treatment was determined by Western blotting of cell lysates prepared by washing twice with cold PBS and direct lysis in reducing sample buffer. After blotting, membranes were incubated with anti-jararhagin antibodies (for visualization of total jararhagin bound) or with extravidin®-peroxidase (for visualization of only biotinylated jararhagin) (1:1000, Sigma) for 1 h and detected as above described. Cells were cultured as monolayers with or without jararhagin stimulation. At different time points, media were collected and separated (20 μl/lane) on 10% SDS-polyacrylamide gels containing 1 mg/ml bovine gelatin (Sigma). Then gels were washed in 2.5% Triton X-100 for 30 min followed by an overnight incubation in metalloproteinase substrate buffer (50 mm Tris-HCl, pH 8.0, 5 mm CaCl2) (28Howard E.W. Bullen E. Banda M.J. J. Biol. Chem. 1991; 266: 13064-13069Google Scholar). Gels were stained with Coomassie Blue R-250 and then destained in water. Total RNA was isolated by direct lysis of the cells in guanidine thiocyanate followed by phenol-chloroform extraction (29Chomezynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Google Scholar). Total RNA (5 μg) was resolved in formaldehyde/agarose gels, blotted onto Hybond-N+ membranes (Amersham Biosciences), and hybridized with random-primed32P-labeled cDNA probes for MT1-MMP (30Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Google Scholar), MMP-1 (31Angel P. Baumann I. Stein B. Dellus H. Rahmsdorf H.J. Herrlich P. Mol. Cell. Biol. 1987; 7: 2256-2266Google Scholar), and the α2 integrin chain (32Hemler M.E. J. Biol. Chem. 1985; 260: 1524-1526Google Scholar). Fibroblasts showed similar adhesion levels to jararhagin- and type I collagen-coated dishes (Fig.1 A). No significant difference was observed between fibroblast adhesion to jararhagin or 1,10-phenanthroline-inactivated jararhagin. As shown in Fig.1 B, cell adhesion to jararhagin was reduced by ∼30% in the presence of blocking antibodies directed against the α2 or β1 integrin subunits; whereas no inhibition was noticed with other function-blocking antibodies directed against α1 or α3 integrins, which both can serve as collagen receptors (2Hynes R.O. Cell. 1992; 9: 11-25Google Scholar). Using a combination of both the α2 and β1 antibodies, cell adhesion to jararhagin was inhibited by up to ∼60% in a dose-dependent manner. When fibroblasts were pre-treated with jararhagin followed by incubation of the cells within collagen lattices there was a notable delay of lattice contraction (Fig. 2). At 24 h untreated fibroblasts contracted the gels to ∼30% of the initial surface area. However, in the presence of 100 nmjararhagin, similar levels of contraction were not observed until 48 h. Cell viability was comparable in treated and untreated fibroblast cultures. Because contraction of fibrillar collagen type I lattices has been shown to be mediated by the integrin α2β1 (12Chan B.M. Matsuura N. Takada Y. Zetter B.R. Hemler M.E. Science. 1991; 251: 1600-1602Google Scholar, 13Klein C.E. Dressel D. Steinmayer T. Mauch C. Eckes B. Krieg T. Bankert R.B. Weber L. J. Cell Biol. 1991; 115: 1427-1436Google Scholar, 14Langholz O. Roeckel D. Mauch C. Kozlowska E. Bank I. Krieg T. Eckes B. J. Cell Biol. 1995; 131: 1903-1915Google Scholar), these results suggest that jararhagin delays the contraction by interfering with the α2β1-collagen interaction. Previous studies have shown that binding of the α2β1 integrin on platelets by PIII snake venom metalloproteinases results in an inhibition of the signaling events normally induced in collagen-stimulated platelets coupled with a potent inhibition of platelet aggregation (22Kamiguti A.S. Markland F.S. Zhou Q. Laing G.D. Theakston R.D.G. Zuzel M. J. Biol. Chem. 1997; 272: 32599-32605Google Scholar, 23De Luca M. Ward C.M. Ohmori K. Andrews R.K. Berndt M.C. Biochem. Biophys. Res. Commun. 1995; 206: 570-576Google Scholar). To determine whether jararhagin binds to cell surface proteins, both supernatants and crude fibroblast membranes were analyzed by SDS-PAGE after 24 and 48 h of incubation with jararhagin (Fig.3). Immunoblotting using a jararhagin-specific antibody detected a band of 55 kDa indicating the presence of jararhagin in the cell culture supernatants. An additional band of ∼33 kDa likely represents the proteolytic degradation fragment of jararhagin comprising the disintegrin-like/cysteine-rich domains (jararhagin-C) (Fig. 3 A). In membranes isolated at 24 h, a weakly stained 55-kDa band corresponding to intact jararhagin was detected. The intensity of this band was significantly increased at 48 h (Fig. 3 B). The additional band of 52 kDa might represent unreduced jararhagin or a proteolytically processed form (13Klein C.E. Dressel D. Steinmayer T. Mauch C. Eckes B. Krieg T. Bankert R.B. Weber L. J. Cell Biol. 1991; 115: 1427-1436Google Scholar). Additionally, these data suggest that jararhagin binding to cell surface proteins on fibroblasts offers protection from proteolytic degradation at the site producing jararhagin-C. An assay was performed to demonstrate the specificity of binding of jararhagin to the cell surface. In these experiments, unlabeled jararhagin was used to displace biotinylated jararhagin from the cell surface of fibroblasts. As shown in Fig.4 A, unlabeled jararhagin is able to displace the cell-surface-associated biotinylated jararhagin in a concentration-dependent manner. In Fig. 4 B, the soluble ectodomain of the integrin α2β1 binds to immobilized jararhagin. However, in contrast to collagen type I, the binding of the soluble α2β1 receptor to jararhagin did not seem to be dependent upon the presence of divalent cations. This suggests the possibility of one or more different binding sites for jararhagin than that for collagen on the receptor molecule. After 24 and 48 h of growth in collagen lattices, total RNA was isolated from fibroblasts pre-treated with 100 nmjararhagin, and the transcript levels for MMP-1 and MT1-MMP were assessed by Northern blot analysis (Fig.5). Control fibroblasts grown in collagen gels showed increased transcript levels for MT1-MMP and MMP-1 at 24 h with a further increase at 48 h culture. At both time points, pre-treatment with jararhagin did not result in significant differences of these transcript levels from those observed in the untreated cells. In addition, there was a similar increase in integrin α2 mRNA level from both untreated and jararhagin-treated fibroblasts. Therefore, pre-treatment of fibroblasts with jararhagin had no apparent effect on fibroblasts grown within collagen lattices. Analysis of fibroblast cell morphology following treatment with increasing concentrations of jararhagin showed a characteristic elongated shape with protrusions of cell extensions identical to that reported for fibroblasts grown in collagen gels (9Mauch C. Hatamochi A. Scharffetter K. Krieg T. Exp. Cell Res. 1988; 178: 493-503Google Scholar). Untreated fibroblasts maintained their characteristic spindle-like morphology with a flattened cell shape (Fig.6). In contrast to fibroblast growth in collagen lattices, in which no significant alterations could be detected, in monolayer cultures treatment with jararhagin produced significant differences as shown in Fig. 5. In monolayer cultures, only very low levels of MT1-MMP and MMP-1 transcripts were observed. However, fibroblasts pre-treated with jararhagin displayed a strong induction of MT1-MMP and MMP-1 mRNA expression together with increased α2-integrin transcript levels. These increases were apparent at 24 h, and by 48 h the increases were comparable to those obtained with fibroblasts cultured within collagen lattices. In addition, the induction of MMP mRNA levels was found to be concentration-dependent, showing maximal stimulation when the cells were pre-treated with 200 nm jararhagin (Fig. 7). To test whether the metalloproteinase activity of jararhagin is required for the induction of MMP-1 and MT1-MMP expression, monolayer cultures of fibroblasts were treated with active or 1,10-phenanthroline-inactivated jararhagin (22Kamiguti A.S. Markland F.S. Zhou Q. Laing G.D. Theakston R.D.G. Zuzel M. J. Biol. Chem. 1997; 272: 32599-32605Google Scholar). As shown in Fig.8, treatment of fibroblasts with proteolytically inactive jararhagin resulted in no significant differences in MMP-1 and MT1-MMP mRNA levels when compared with fibroblasts treated with active jararhagin. The apparent slight reduction of the mRNA levels observed after treatment with inactivated jararhagin was due to the presence of the general metalloproteinase inhibitor 1,10-phenanthroline as shown by the control fibroblast treatment with 1,10-phenanthroline. Western blot analysis of MT1-MMP in crude membrane preparations from cells treated with jararhagin displayed increased levels of the active 60-kDa MT1-MMP protein form at 24 h as well as at 48 h as compared with untreated fibroblasts (Fig. 9 A). A low level of an additional immunoreactive protein of 63-kDa band corresponding to the unprocessed zymogen was also detected (33Lehti K. Lohi J. Valtanen H. Keski-Oja J. Biochem. J. 1998; 334: 345-353Google Scholar, 34Kurschat P. Zigrino P. Nischt R. Breitkopf K. Steurer P. Klein E.C. Krieg T. Mauch C. J. Biol. Chem. 1999; 274: 21056-21062Google Scholar). This increase in MT1-MMP production in treated monolayer culture was paralleled by enhanced pro-MMP-2 activation as indicated by the appearance of the 62/59-kDa forms correspondent to the active enzyme (Fig. 9 B). Analysis of MMP-1 protein in supernatants of cells treated with jararhagin showed increased inactive MMP-1 forms, 52/57 kDa, as well as appearance of active MMP-1, 42/47 kDa, at both 24 and 48 h treatment (Fig. 9 C). We also assessed the possibility that the proteolytic activity of jararhagin might be directly involved in MT1-MMP and pro-MMP-2 activation. Treatment of cell membranes, prepared from an MT1-MMP-stable expressing cell line containing only the pro-form of MT1-MMP (34Kurschat P. Zigrino P. Nischt R. Breitkopf K. Steurer P. Klein E.C. Krieg T. Mauch C. J. Biol. Chem. 1999; 274: 21056-21062Google Scholar) with jararhagin, did not result in activation of pro-MT1-MMP (data not shown). In addition, treatment of media containing pro-MMP-2 with jararhagin also failed to show activation of the zymogen (data not shown). These observations suggest that neither the activation of pro-MT1-MMP nor the activation of pro-MMP-2 was a result of the proteolytic activity of jararhagin. As shown above, jararhagin did not block the α2β1-induced MMP-1 and MT1-MMP up-regulation in fibroblasts grown in collagen lattices. Surprisingly, in monolayer cultures, the addition of jararhagin resulted in increased transcript levels for the α2-integrin subunit and MMP-1 and MT1-MMP indicating an activation rather than inhibition of this integrin receptor. It has been demonstrated that fibroblasts grown in collagen lattices respond with an up-regulation the α2β1 integrin expression, whereas there is no up-regulation when fibroblasts are grown as monolayers (16Zigrino P. Drescher C. Mauch C. Eur. J. Cell Biol. 2001; 80: 68-77Google Scholar). Treatment of fibroblasts with jararhagin followed by growth as monolayers caused a significant increase of α2β1-integrin immunostaining, whereas no specific staining was detected in untreated monolayer cultures (Fig.10 A). Western blot analysis of the α2 integrin subunit in cells treated with jararhagin displayed slightly increased protein levels at 24 h followed by a significant increase at 48 h treatment (Fig.10 B). The observed increase in α2 integrin levels displayed on the cell surface may
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